This invention relates generally to compositions of cyanine dye bioconjugates with bioactive molecules for diagnosis and therapy, and particularly for visualization and detection of tumors.
Several dyes that absorb and emit light in the visible and near-infrared region of electromagnetic spectrum are currently being used for various biomedical applications due to their biocompatibility, high molar absorptivity, and/or high fluorescence quantum yields. The high sensitivity of the optical modality in conjunction with dyes as contrast agents parallels that of nuclear medicine, and permits visualization of organs and tissues without the undesirable effect of ionizing radiation.
Cyanine dyes with intense absorption and emission in the near-infrared (NIR) region are particularly useful because biological tissues are optically transparent in this region (B. C. Wilson, Optical properties of tissues. Encyclopedia of Human Biology, 1991, 5, 587-597). For example, indocyanine green, which absorbs and emits in the NIR region, has been used for monitoring cardiac output, hepatic functions, and liver blood flow (Y-L. He, et al., Measurement of blood volume using indocyanine green measured with pulse-spectrometry: Its reproducibility and reliability. Critical Care Medicine, 1998, 26(8), 1446-1451; J. Caesar, et al., The use of Indocyanine green in the measurement of hepatic blood flow and as a test of hepatic function. Clin. Sci. 1961, 21, 43-57), and its functionalized derivatives have been used to conjugate biomolecules for diagnostic purposes (R. B. Mujumdar, et al., Cyanine dye labeling reagents: Sulformdocyanine succinimidyl esters. Bioconjugate Chemistry, 1993, 4(2), 105-111; U.S. Pat. No. 5,453,505; WO 98/48846; WO 98/22146; WO 96/17628; WO 98/48838).
A major drawback in the use of cyanine dye derivatives is the potential for hepatobiliary toxicity resulting from the rapid clearance of these dyes by the liver (G. R. Cherrick, et al., Indocyanine green: Observations on its physical properties, plasma decay, and hepatic extraction. J. Clinical Investigation, 1960, 39, 592-600). This is associated with the tendency of cyanine dyes in solution to form aggregates, which could be taken up by Kupffer cells in the liver.
Various attempts to obviate this problem have not been very successful. Typically, hydrophilic peptides, polyethyleneglycol or oligosaccharide conjugates have been used, but these resulted in long-circulating products, which are eventually still cleared by the liver. Another major difficulty with current cyanine and indocyanine dye systems is that they offer a limited scope in the ability to induce large changes in the absorption and emission properties of these dyes. Attempts have been made to incorporate various heteroatoms and cyclic moieties into the polyene chain of these dyes (L. Strekowski, et al., Substitution reactions of a nucleofugal group in hetamethine cyanine dyes. J. Org. Chem., 1992, 57, 4578-4580; N. Narayanan, and G. Patonay, A new method for the synthesis of heptamethine cyanine dyes: Synthesis of new near infrared fluorescent labels. J. Org. Chem., 1995, 60, 2391-2395; U.S. Pat. Nos. 5,732,104; 5,672,333; and 5,709,845), but the resulting dye systems do not show large differences in absorption and emission maxima, especially beyond 830 nm where photoacoustic diagnostic applications are very sensitive. They also possess a prominent hydrophobic core, which enhances liver uptake. Further, most cyanine dyes do not have the capacity to form starburst dendrimers, which are useful in biomedical applications.
For the purpose of tumor detection, many conventional dyes are useful for in vitro applications because of their highly toxic effect on both normal and abnormal tissues. Other dyes lack specificity for particular organs or tissues and, hence, these dyes must be attached to bioactive carriers such as proteins, peptides, carbohydrates, and the like to deliver the dyes to specific regions in the body. Several studies on the use of near infrared dyes and dye-biomolecule conjugates have been published (G. Patonay and M. D. Antoine, Near-infrared Fluorogenic Labels: New Approach to an Old Problem, Analytical Chemistry, 1991, 63:321A-327A and references therein; M. Brinkley, A Brief Survey of Methods for Preparing Protein Conjugates with Dyes, Haptens, and Cross-Linking Reagents, Perspectives in Bioconjugate Chemistry 1993, pp. 59-70, C. Meares (Ed), ACS Publication, Washington, D.C.; J. Slavik, Fluorescent Probes in Cellular and Molecular Biology, 1994, CRC Press, Inc.; U.S. Pat. No. 5,453,505; WO 98/48846; WO 98/22146; WO 96/17628; WO 98/48838). Of particular interest is the targeting of tumor cells with antibodies or other large protein carriers such as transferrin as delivery vehicles (A. Becker, et al., xe2x80x9cTransferrin Mediated Tumor Delivery of Contrast Media for Optical Imaging and Magnetic Resonance Imagingxe2x80x9d, Biomedical Optics meeting, Jan. 23-29, 1999, San Jose, Calif.). Such an approach has been widely used in nuclear medicine applications. Its major advantage is the retention of a carrier""s tissue specificity, since the molecular volume of the dye is substantially smaller than the carrier. However, this approach does have some serious limitations in that the diffusion of high molecular weight bioconjugates to tumor cells is highly unfavorable, and is further complicated by the net positive pressure in solid tumors (R. K. Jain, Barriers to Drug Delivery in Solid Tumors, Scientific American 1994, 271:58-65. Furthermore, many dyes in general, and cyanine dyes, in particular, tend to form aggregates in aqueous media that lead to fluorescence quenching.
Therefore, there is a need for dyes that could prevent dye aggregation in solution, that are predisposed to form dendrimers, that are capable of absorbing or emitting beyond 800 nm, that possess desirable photophysical properties, and that are endowed with tissue-specific targeting capability.
The invention is directed to compositions, and methods of preparing the compositions, of low molecular weight biomolecule-dye conjugates to enhance tumor detection. The inventive compositions preserve the fluorescence efficiency of the dye molecules, do not aggregate in solution, form starburst dendrimers, are capable of absorbing or omitting light in the near infrared region (beyond 800 mm), and can be rendered tissue-specific.
In one embodiment, the inventive composition comprises cyanine dyes of general formula 1.
wherein W3 and X3 may be the same or different and are selected from the group consisting of xe2x80x94CR1R2, xe2x80x94Oxe2x80x94, xe2x80x94NR3, xe2x80x94Sxe2x80x94, and xe2x80x94Se; Y3 is selected from the group consisting of xe2x80x94(CH2)axe2x80x94CONHxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94CONHxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94NHCOxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94NHCOxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94N(R3)xe2x80x94(CH2)bxe2x80x94CONHxe2x80x94Bm, (CH2)axe2x80x94N(R3)xe2x80x94(CH2)cxe2x80x94NHCOxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94CONHxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94NHCOxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94(CH2)axe2x80x94CONHxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94(CH2)axe2x80x94NHCOxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)dxe2x80x94CONHxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)dxe2x80x94NHCOxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94NR3R4, and xe2x80x94CH2(CH2OCH2)bxe2x80x94CH2NR3R4; Z3 is selected from the group consisting of xe2x80x94(CH2)axe2x80x94CONHxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94CONHxe2x80x94Dm, xe2x80x94(CH2)axe2x80x94NHCOxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94NHCOxe2x80x94Dm, xe2x80x94(CH2)axe2x80x94N(R3)xe2x80x94(CH2)bxe2x80x94CONHxe2x80x94Dm, (CH2)axe2x80x94N(R3)xe2x80x94(CH2)cxe2x80x94NHCOxe2x80x94Dm, xe2x80x94(CH2)axe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94CONHxe2x80x94Dm, xe2x80x94(CH2)axe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94NHCOxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94(CH2)axe2x80x94CONHxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94(CH2)axe2x80x94NHCOxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)dxe2x80x94CONHxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)dxe2x80x94NHCOxe2x80x94Dm, xe2x80x94(CH2)axe2x80x94NR3R4, and xe2x80x94CH2(CH2OCH2)bxe2x80x94CH2NR3R4; A1 is a single or a double bond; B1, C1, and D1 may the same or different and are selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Sexe2x80x94, xe2x80x94Pxe2x80x94, xe2x80x94CR1R2, xe2x80x94CR1, alkyl, NR3, and xe2x80x94Cxe2x95x90O; A1, B1, C1, and D1 may together form a 6- to 12-membered carbocyclic ring or a 6- to 12-membered heterocyclic ring optionally containing one or more oxygen, nitrogen, or sulfur atom; a3 and b3 independently vary from 0 to 5; R1 to R4, and R29 to R37 are independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, cyano, nitro, halogen, saccharide, peptide, xe2x80x94CH2(CH2OCH2)bxe2x80x94CH2xe2x80x94OH, xe2x80x94(CH2)axe2x80x94CO2H, xe2x80x94(CH2)axe2x80x94CONHxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94CONHxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94NHCOxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94NHCOxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94OH and xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CO2H; Bm and Dm are independently selected from the group consisting of a bioactive peptide, a protein, a cell, an antibody, an antibody fragment, a saccharide, a glycopeptide, a peptidomimetic, a drug, a drug mimic, a hormone, a metal chelating agent, a radioactive or nonradioactive metal complex, and an echogenic agent; a and c are independently from 1 to 20; and b and d are independently from 1 to 100.
In a second embodiment, the inventive composition comprises cyanine dyes of general formula 2. 
wherein W4 and X4 may be the same or different and are selected from the group consisting of xe2x80x94CR1R2, xe2x80x94Oxe2x80x94, xe2x80x94NR3, xe2x80x94Sxe2x80x94, and xe2x80x94Se; Y4 is selected from the group consisting of xe2x80x94(CH2)axe2x80x94CONHxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94CONHxe2x80x94Bm, xe2x80x94(CH2)xe2x80x94NHCOxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94NHCOxe2x80x94Bm, (CH2)axe2x80x94N(R3)xe2x80x94(CH2)bxe2x80x94CONHxe2x80x94Bm, (CH2)axe2x80x94N(R3)xe2x80x94(CH2)cxe2x80x94NHCOxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94CONHxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94NHCOxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94(CH2)axe2x80x94CONHxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94(CH2)axe2x80x94NHCOxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)dxe2x80x94CONHxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)dxe2x80x94NHCOxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94NR3R4, and xe2x80x94CH2(CH2OCH2)bxe2x80x94CH2NR3R4; Z4 is selected from the group consisting of xe2x80x94(CH2)xe2x80x94CONHxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94CONHxe2x80x94Dm, xe2x80x94(CH2)axe2x80x94NHCOxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94NHCOxe2x80x94Dm, xe2x80x94(CH2)axe2x80x94N(R3)xe2x80x94(CH2)bxe2x80x94CONHxe2x80x94Dm, (CH2)axe2x80x94N(R3)xe2x80x94(CH2)cxe2x80x94NHCOxe2x80x94Dm, xe2x80x94(CH2)axe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94CONHxe2x80x94Dm, xe2x80x94(CH2)axe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94NHCOxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94(CH2)axe2x80x94CONHxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94(CH2)axe2x80x94NHCOxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)dxe2x80x94CONHxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)dxe2x80x94NHCOxe2x80x94Dm, xe2x80x94(CH2)axe2x80x94NR3R4, and xe2x80x94CH2(CH2OCH2)bxe2x80x94CH2NR3R4; A2 is a single or a double bond; B2, C2, and D2 may be the same or different and are selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Sexe2x80x94, xe2x80x94Pxe2x80x94, xe2x80x94CR1R2, xe2x80x94CR1, alkyl, NR3, and xe2x80x94Cxe2x95x90O; A2, B2, C2, and D2 may together form a 6- to 12-membered carbocyclic ring or a 6- to 12-membered heterocyclic ring optionally containing one or more oxygen, nitrogen, or sulfur atom; a4 and b4 independently vary from 0 to 5; R1 to R4, and R45 to R57 are independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, cyano, nitro, halogen, saccharide, peptide, xe2x80x94CH2(CH2OCH2)bxe2x80x94CH2xe2x80x94OH, xe2x80x94(CH2)axe2x80x94CO2H, xe2x80x94(CH2)axe2x80x94CONHxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94CONHxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94NHCOxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94NHCOxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94OH and xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CO2H; Bm and Dm are independently selected from the group consisting of a bioactive peptide, a protein, a cell, an antibody, an antibody fragment, a saccharide, a glycopeptide, a peptidomimetic, a drug, a drug mimic, a hormone, a metal chelating agent, a radioactive or nonradioactive metal complex, and an echogenic agent; a and c are independently from 1 to 20; and b and d are independently from 1 to 100.
In a third embodiment, the inventive composition comprises cyanine dyes of general formula 3
wherein W5 and X5 may be the same or different and are selected from the group consisting of xe2x80x94CR1R2, xe2x80x94Oxe2x80x94, xe2x80x94NR3, xe2x80x94Sxe2x80x94, and xe2x80x94Se; Y5 is selected from the group consisting of xe2x80x94(CH2)axe2x80x94CONHxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94CONHxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94NHCOxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94NHCOxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94N(R3)xe2x80x94(CH2)bxe2x80x94CONHxe2x80x94Bm, (CH2)axe2x80x94N(R3)xe2x80x94(CH2)cxe2x80x94NHCOxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94N(R3)xe2x80x94(CH2)bxe2x80x94CH2xe2x80x94CONHxe2x80x94Bm, xe2x80x94(CH2), xe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94NHCOxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94(CH2)axe2x80x94CONHxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94(CH2)axe2x80x94NHCOxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)dxe2x80x94CONHxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)dxe2x80x94NHCOxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94NR3R4, and xe2x80x94CH2(CH2OCH2)bxe2x80x94CH2NR3R4; Z5 is selected from the group consisting of xe2x80x94(CH2)axe2x80x94CONHxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94CONHxe2x80x94Dm, xe2x80x94(CH2)axe2x80x94NHCOxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94NHCOxe2x80x94Dm, xe2x80x94(CH2)axe2x80x94N(R3)xe2x80x94(CH2)bxe2x80x94CONHxe2x80x94Dm, (CH2)axe2x80x94N(R3)xe2x80x94(CH2)cxe2x80x94NHCOxe2x80x94Dm, xe2x80x94(CH2)xe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94CONHxe2x80x94Dm, xe2x80x94(CH2)axe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94NHCOxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94(CH2)axe2x80x94CONHxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94(CH2)axe2x80x94NHCOxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)dxe2x80x94CONHxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)dxe2x80x94NHCOxe2x80x94Dm, xe2x80x94(CH2)axe2x80x94NR3R4, and xe2x80x94CH2(CH2OCH2)bxe2x80x94CH2NR3R4; A3 is a single or a double bond; B3, C3, and D3 may be the same or different and are selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Sexe2x80x94, xe2x80x94Pxe2x80x94, xe2x80x94CR1R2, xe2x80x94CR1, alkyl, NR3, and xe2x80x94Cxe2x95x90O; A3, B3, C3, and D3 may together form a 6- to 12-membered carbocyclic ring or a 6- to 12-membered heterocyclic ring optionally containing one or more oxygen, nitrogen, or sulfur atom; a5 is independently from 0 to 5; R1 to R4, and R58 to R66 are independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, cyano, nitro, halogen, saccharide, peptide, xe2x80x94CH2(CH2OCH2)bxe2x80x94CH2xe2x80x94OH, xe2x80x94(CH2)axe2x80x94CO2H, xe2x80x94(CH2)axe2x80x94CONHxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94CONHxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94NHCOxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94NHCOxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94OH and xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CO2H; Bm and Dm are independently selected from the group consisting of a bioactive peptide, a protein, a cell, an antibody, an antibody fragment, a saccharide, a glycopeptide, a peptidomimetic, a drug, a drug mimic, a hormone, a metal chelating agent, a radioactive or nonradioactive metal complex, and an echogenic agent; a and c are independently from 1 to 20; and b and d are independently from 1 to 100.
In a fourth embodiment, the inventive composition comprises cyanine dyes of general formula 4
wherein W6 and X6 may be the same or different and are selected from the group consisting of xe2x80x94CR1R2, xe2x80x94Oxe2x80x94, xe2x80x94NR3, xe2x80x94Sxe2x80x94, and xe2x80x94Se; Y6 is selected from the group consisting of xe2x80x94(CH2)axe2x80x94CONHxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94CONHxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94NHCOxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94NHCOxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94N(R3)xe2x80x94(CH2)bxe2x80x94CONHxe2x80x94Bm, (CH2)axe2x80x94N(R3)xe2x80x94(CH2)cxe2x80x94NHCOxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94CONHxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94NHCOxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94(CH2)a-CONHxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94(CH2)axe2x80x94NHCOxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2CH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)dxe2x80x94CONHxe2x80x94Bm, CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)dxe2x80x94NHCOxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94NR3R4, and xe2x80x94CH2(CH2CH2)bxe2x80x94CH2NR3R4; Z6 is selected from the group consisting of xe2x80x94(CH2)axe2x80x94CONHxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94CONHxe2x80x94Dm, xe2x80x94(CH2)axe2x80x94NHCOxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94NHCOxe2x80x94Dm, xe2x80x94(CH2)axe2x80x94N(R3)xe2x80x94(CH2)bxe2x80x94CONHxe2x80x94Dm, (CH2)axe2x80x94N(R3)xe2x80x94(CH2)cxe2x80x94NHCOxe2x80x94Dm, xe2x80x94(CH2)xe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94CONHxe2x80x94Dm, xe2x80x94(CH2)axe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94NHCOxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94(CH2)axe2x80x94CONHxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94(CH2)axe2x80x94NHCOxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)dxe2x80x94CONHxe2x80x94Dm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94N(R3)xe2x80x94CH2xe2x80x94(CH2OCH2)dxe2x80x94NHCOxe2x80x94Dm, xe2x80x94(CH2)axe2x80x94NR3R4, and xe2x80x94CH2(CH2OCH2)bxe2x80x94CH2NR3R4; A4 is a single or a double bond; B4, C4, and D4 may be the same or different and are selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Sexe2x80x94, xe2x80x94Pxe2x80x94, xe2x80x94CR1R2, xe2x80x94CR1, alkyl, NR3, and xe2x80x94Cxe2x95x90O; A4, B4, C4, and D4 may together form a 6- to 12-membered carbocyclic ring or a 6- to 12-membered heterocyclic ring optionally containing one or more oxygen, nitrogen, or sulfur atom; a6 is independently from 0 to 5; R1 to R4, and R67 to R79 are independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, cyano, nitro, halogen, saccharide, peptide, xe2x80x94CH2(CH2OCH2)bxe2x80x94CH2xe2x80x94OH, xe2x80x94(CH2)axe2x80x94CO2H, xe2x80x94(CH2)axe2x80x94CONHxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94CONHxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94NHCOxe2x80x94Bm, xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CH2xe2x80x94NHCOxe2x80x94Bm, xe2x80x94(CH2)axe2x80x94OH or xe2x80x94CH2xe2x80x94(CH2OCH2)bxe2x80x94CO2H; Bm and Dm are independently selected from the group consisting of a bioactive peptide, a protein, a cell, an antibody, an antibody fragment, a saccharide, a glycopeptide, a peptidomimetic, a drug, a drug mimic, a hormone, a metal chelating agent, a radioactive or nonradioactive metal complex, and an echogenic agent; a and c are independently from 1 to 20; and b and d are independently from 1 to 100.
The invention will be further appreciated in light of the following figures, detailed description, and examples.